Project Summary/Abstract Resiquimod gel is a topically applied immunomodulator and TLR7/8 agonist. It stimulates dendritic cells (DC) in both healthy and inflamed skin and can potently enhance immune responses. In a small phase I trial of in 12 heavily pretreated patients with refractory, skin limited cutaneous T cell lymphoma (CTCL), topical resiquimod reversed immune evasion, improved immunodetection and induced effector anticancer responses both locally and systemically. 90% of patients had reduction in the malignant T cell clone in treated lesions and 83% had regression of both treated and distant, untreated skin lesions, demonstrating that this topical therapy enhances systemic immune responses. The ability of this drug to enhance both local and systemic immune responses suggests that topical resiquimod could be a promising new treatment for both skin infections and cancer. We propose time sensitive, first in human ancillary studies of fresh biopsy specimens and blood samples from an industry sponsored, multicenter, phase II, double blind, placebo-controlled trial of resiquimod gel in the treatment of stage IA, IB and IIA CTCL. We hypothesize that topical resiquimod enhances both innate and adaptive immune responses and we propose three Aims to study the effects of topical resiquimod on i) the innate immune system, ii) the adaptive immune system, and iii) to discover the mechanisms by which untreated lesions regress. We will use single cell RNA sequencing (scRNA-seq), NanoString based gene expression profiling, multiplex immunostaining and TCR sequencing to study skin biopsies before and after resiquimod therapy and we will use cytometry by time of flight (CyTOF), TCR sequencing and functional assays to study blood samples before and after treatment. We will evaluate specific hypotheses that resiquimod induces a shift in macrophage polarization from M2 to M1, activates NK cells, enhances recruitment of T cells into skin, reverses exhaustion of T cells within the tumor microenvironment and/or generates new tumor-specific T cells. We will determine if regression of untreated lesions is associated with detectable levels of type I interferons or Th1 cytokines, activation of circulating DC or NK cells, or travel of tumor-specific T cells through the bloodstream to untreated lesions. These studies are time sensitive because they require the use of fresh biopsy specimens from an ongoing clinical trial. Our studies will clarify the mechanisms of action of this promising new medication and determine how it reverses immune evasion and enhances immunodetection in CTCL. These studies may support the use of resiquimod in the treatment of other skin cancers and infections.